1. Field
The disclosed methods and apparatus relate to the analysis of analytes including, but not limited to, proteins, polypeptides, peptides, lipids and polysaccharides. In particular, the methods and apparatus relates to protein, polypeptide and/or peptide identification and/or sequencing.
2. Related Art
Identification and/or sequencing of analytes, such as proteins, are critical for medical diagnostics, forensics, toxicology, pathology, biological warfare, public health and numerous other fields. The ability to identify a particular pathogen or agent may depend on identification of one or more specific analytes characteristic of that pathogen or agent. Identification of regulatory pathways involved in disease processes, metabolism, growth and cell division may depend on identification and/or sequencing of analytes. Although a great deal of research is presently directed towards identification and/or sequencing of nucleic acids or proteins, other analytes such as carbohydrates, polysaccharides, lipids, fatty acids, etc. may be of importance. The methods and apparatus disclosed herein are focused on identification and/or sequencing of proteins, polypeptides and peptides. However, they are also of use for analysis of other types of analytes.
Existing methods for protein sequencing, based on the Edman degradation technique, are limited by the length of the protein that can be sequenced. Accurate sequence determination is limited to about 50 to 100 amino acid residues per sequencing run. Sequencing of longer proteins, which may be thousands of amino acid residues in length, requires cleavage into smaller fragments and assembly of overlapping short sequences. The process is laborious, expensive, inefficient and time-consuming and typically requires the use of radioactive labels and other hazardous chemicals, which can pose safety and waste disposal problems.
A variety of techniques are available for identification of proteins, polypeptides and peptides. Commonly, these involve binding and detection of antibodies that can recognize one or more epitopic domains on the protein. Although antibody-based identification of proteins is fairly rapid, such assays may occasionally show unacceptably high levels of false positive or false negative results, due to cross-reactivity of the antibody with different antigens, low antigenicity of the target analyte (leading to low sensitivity of the assay), non-specific binding of antibody to various surfaces, etc. They also require the preparation of antibodies that can recognize an individual protein or peptide. As such, they are not suitable for the identification of novel proteins that have not previously been characterized. More recently, mass spectroscopy has been used for peptide identification and/or sequencing. Proteins and polypeptides may be cleaved into smaller fragments and the amino acid composition of the fragments may be identified by mass spectroscopy. Analysis of a sufficient number of overlapping fragments can provide data on amino acid sequence. This process is also laborious, expensive and requires substantial purification of the protein or peptide to be analyzed.
A need exists in the art for methods and apparatus suitable for the identification and/or sequencing of analytes, including proteins and peptides that have not previously been identified or characterized.